Expression of costimulatory molecules in human neuroblastoma. Evidence that CD40+ neuroblastoma cells undergo apoptosis following interaction with CD40L.

Tumour cells display low to absent expression of costimulatory molecules. Here, we have investigated the expression of costimulatory molecules (CD40, CD80, CD86, PD-1L, B7H2, OX40L and 4-1BBL) in human neuroblastoma (NB) cells, since virtually no information is available on this issue. Both established NB cell lines and primary tumours were tested by RT-PCR and flow cytometry. Neuroblastoma cell lines expressed the transcripts of all costimulatory molecule genes, but not the corresponding proteins. Culture of NB cell lines with human recombinant (r)IFN-gamma induced surface expression of CD40 in half of them. Primary NB cells showed CD40, CD80, CD86, OX40L, 4-1BBL, but not PD-1L and B7H2, mRNA expression. Surface CD40 was consistently detected on primary NB cells by flow cytometry. Interferon-gamma gene-transfected NB cells expressed constitutively surface CD40 and were induced into apoptosis by incubation with rCD40L through a caspase-8-dependent mechanism. CD40 may represent a novel therapeutic target in NB.

Lack of HLA-class I antigens in human neuroblastoma cells: analysis of its relationship to TAP and tapasin expression.

We studied the constitutive and the interferon (IFN)-gamma-induced expression of HLA class I antigen heavy chain, beta2-microglobulin (beta2m), TAP-1, TAP-2 and tapasin in a panel of eleven neuroblastoma cell lines. Surface expression of HLA class I antigens was low in eight out of eight neuroblastoma cell lines bearing MYC-N amplification and/or 1p deletion, while two out of three neuroblastoma cell lines lacking these genetic alterations showed normal expression. IFN-gamma treatment restored HLA class I antigen surface expression in all neuroblastoma cell lines. Eight out of 11 neuroblastoma cell lines did not express TAP-1 mRNA and three of them also lacked TAP-2 mRNA. beta2 m mRNA was barely detectable or absent in five neuroblastoma cell lines, while tapasin mRNA was always expressed. IFN-gamma upregulated the expression of HLA class I heavy chain, beta2 m, TAP-1, TAP-2 and tapasin, as detected at mRNA or protein level. Post-transcriptional events were involved in altered TAP-1 and beta2 m expression in one peculiar neuroblastoma cell line. These data indicate that multiple mechanisms play a role in the HLA class I antigen-deficient phenotype of human neuroblastoma.

Characterization and tumorigenicity of human neuroblastoma cells transfected with the IL-2 gene.

Immunization of cancer patients with cytokine-engineered tumor cells is being currently tested in several trials. To test the feasibility of this approach in neuroblastoma (NB) patients we investigated the functional consequences of interleukin-2 (IL-2) gene transfer into NB cell lines. Two human NB cell lines were transfected with the plasmid expression vector RSV.5neo containing the human IL-2 cDNA, and their tumorigenicity was evaluated in a nude mice xenograft model after characterization of the growth patterns and phenotypic features in vitro. The combination of IL-2 gene transfection and the xenograft model in nude mice was chosen on the basis of the low or absent expression of HLA class I antigen in human NB tumors. Our aim was to evaluate the effectiveness of an immunization protocol that could elicit a nonspecific antitumor response. The IL-2 stable transfectants were morphologically identical to parental or vector-transfected cells but completely lost tumorigenicity and inhibited, through a bystander effect, the growth of parental cells injected simultaneously at the same site. Histologic and immunohistochemical analysis of the nodules showed extensive necrosis with severe endothelial damage. The infiltrating cells were mainly macrophages, while natural killer (NK) cells were scarce. However, depletion of NK cells by anti-CD122 monoclonal antibody indicated that the rejection process required NK cell activity. The relevance of these data for the development of therapeutic approaches using cytokine-engineered NB cell lines is discussed.

Expression of MAGE-1, MAGE-3 and MART-1 genes in neuroblastoma.

The human MAGE-1, MAGE-3 and MART-1 genes code for antigens that are specifically recognized by cytolytic T lymphocytes in a MHC-restricted manner. The MAGE-1 and MAGE-3 genes are expressed in tumors of different histotypes but not in normal adult tissues (with the exception of testis), while the MART-1 gene appears to be selectively expressed in melanoma. MAGE-1, MAGE-3 and MART-1 antigens may therefore constitute useful targets for specific anti-tumor immunization of cancer patients. Here we have investigated the expression of MAGE-1, MAGE-3 and MART-1 in 11 neuroblastoma (NB) cell lines and 73 NB tumor masses. MAGE-1 and MAGE-3 transcripts were detected simultaneously in 36% of the cell lines and in 16% of tumor samples. The MAGE-1 gene was never expressed alone except in one tumor. In contrast, MAGE-3 mRNA was found in approximately 40% of the NB tumor samples in the absence of MAGE-1 mRNA. No expression of the MART-1 gene was observed in any cell line or tumor sample. No correlation was found between MAGE gene expression and clinical stage, event-free survival and presence or absence of N-myc amplification.